1. Field of the Invention
The present invention relates to the field of recombinant protein production in bacterial hosts. In particular, the present invention relates to a production process for obtaining high levels of soluble recombinant CRM197 protein from E. coli. The invention also relates to purification and characterization methods for CRM197 as well as uses of the CRM197 produced by the method.
2. Description of the Background
Diphtheria toxin (DT) is a proteinaceous exotoxin synthesized and secreted by pathogenic strains of Corynebacterium diphtheriae. These pathogenic strains contain a bacteriophage lysogen that carries the toxin gene. Diphtheria toxin is an ADP-ribosylating enzyme that is secreted as a proenzyme of 535 residues and processed by trypsin-like proteases with release of two fragments (A and B). Fragment A uses NAD as a substrate, catalyzing the cleavage of the N-glycosidic bond between the nicotinamide ring and the N-ribose and mediating the covalent transfer of the ADP-ribose (ADPRT activity) to the modified histidine 715 (diphthamide) of the elongation factor EF-2. This post-translational diphthamide modification inactivates EF-2, halting protein synthesis and resulting in cell death. The A fragment of DT (also named C domain) carries the catalytic active site and is the only fragment of the toxin required for the final step of intoxication. The R domain, carried on the B fragment, mediates binding to receptors on the host cell surface and the T domain, also carried on the B fragment, promotes the pH-dependent transfer of fragment A to the cytoplasm. An Arginine-rich disulfide-linked loop connects fragment A to fragment B (or domain C to domains TR). This inter-chain disulfide bond is the only covalent link between the two fragments after proteolytic cleavage of the chain at position 186. The isolation of various non-toxic and partially toxic immunologically cross-reacting forms of diphtheria toxins (CRMs or cross reacting materials) resulted in discovery of CRM197 (Uchida et al., Journal of Biological Chemistry 248, 3845-3850, 1973; see also Giannini et al. Nucleic Acids Res. 1984 May 25; 12(10):4063-9). Preferably, CRMs can be of any size and composition that contain all or a portion of DT.
CRM197 is a largely enzymatically inactive and nontoxic form of diphtheria toxin that contains a single amino acid substitution G52E. This mutation causes intrinsic flexibility of the active-site loop in front of the NAD-binding site and reduces the ability of CRM197 to bind NAD and eliminates toxic properties of DT (Malito et al., Proc Natl Acad Sci USA 109(14):5229-342012) Like DT, CRM197 has two disulfide bonds. One disulfide joins Cys186 to Cys201, linking fragment A to fragment B. A second disulfide bridge joins Cys461 to Cys471 within fragment B. Both DT and CRM197 have fragment A-associated nuclease activity (Bruce et al., Proc. Natl. Acad. Sci. USA 87, 2995-8, 1990).
Many antigens are poorly immunogenic, especially in infants, unless chemically linked to a protein (“conjugation”), thereby forming a conjugate or conjugate vaccine. The protein component of these conjugate vaccines is also called the “carrier protein”. CRM197 is commonly used as the carrier protein for protein-carbohydrate and hapten-protein conjugates. As a carrier protein, CRM197 has a number of advantages over diptheria toxoid as well as other toxoid proteins, many of which have been documented (Shinefield Vaccine, 28:4335, 2010, Broker et al, Biologicals, 39:195 2011). For example since CRM197 is genetically detoxified, it retains a larger complement of lysines, which are used for conjugation but are blocked by chemical toxoiding. CRM197 has proven to be an effective carrier protein for Streptococcus pneumonia capsular polysaccharides, as evidenced by the success of PREVNAR™ (Pfizer), a vaccine consisting of up to 13 capsular polysaccharides chemically linked to CRM197. There is also evidence suggesting that compared with tetanus toxoid, there is less carrier-induced suppression of the immune response, especially when there are many individual polysaccharides linked to the same carrier protein.
CRM197 and native DT have a similar affinity for the diphtheria toxin receptor (DTR), which has an identical amino acid sequence to the HB-EGF precursor pro-HB-EGF (Mitamura et al., J. Biol. Chem. 272(43):27084-90, 1997). CRM197 binds to the soluble form of HB-EGF, as well as to the membrane form pro-HB-EGF, and inhibits HB-EGF mitotic action by preventing its binding to EGF receptor. Thus CRM197 may also have a future role in cancer therapy (Miyamoto et al., Anticancer Res. November-December 27(6A):3713-21, 2007).
CRM197 has been produced in the original host Corynebacterium, but yields are low, typically <50 mg/L and, in addition, Corynebacterium growth is relatively slow as compared with, for example, E. coli. There are proprietary strains of Corynebacterium that have been engineered to produce CRM197 at higher levels (U.S. Pat. No. 5,614,382). CRM197 has also been expressed in a proprietary strain of Psuedomonas fluorescens and expressed at high levels. Production of CRM197 in E. coli would be advantageous since E. coli is a BL1 level organism that is inexpensive to culture and propagate. Production of CRM197 in E. coli has mainly resulted in insoluble inclusion bodies (generally insoluble), which then requires a difficult refolding process, resulting in low yields (EP20100742260) or with an additional peptide sequence (a tag) (J Biotechnol. 2010 December 20; 156(4):245-52, Overexpression and purification of the recombinant diphtheria toxin variant CRM197 in Escherichia coli. Stefan A, Conti M, Rubboli D, Ravagli L, Presta E, Hochkoeppler A. A method for the overexpression of soluble tag free CRM197 in E. coli suitable for the large quantity protein production, has not been reported. Thus, there is a need for better methods to produce CRM197 in an efficient and cost-effective manner.